A method for improving the induction rate of protocorm and sporophyte of goldthread

Abstract

This invention discloses a method for improving the induction rate of prothallus and sporophyte in *Cibotium barometz*, comprising: S1, sterilizing the substrate with a fungicide, wherein the fungicide includes thiophanate-methyl and tebuconazole, and after sterilization, reducing the substrate moisture content to 40-50%, obtaining a sterilized substrate; S2, contacting the bottom of the substrate with a hormone aqueous solution, obtaining a substrate absorbent with the hormone aqueous solution; S3, sowing *Cibotium barometz* spores on the substrate surface for cultivation. This invention shortens the spore germination time of *Cibotium barometz*, improves the induction rate of prothallus and sporophyte, and is simple and easy to implement. Using this method to cultivate *Cibotium barometz* seedlings, prothallus are induced in an average of 22 days, and sporophyte are induced in 66 days. This method can be applied on a large scale to the sowing and seedling cultivation of *Cibotium barometz*, saving seedling time and labor costs.

Description

Technical Field

[0001] This invention relates to the field of seedling propagation technology, specifically to a method for improving the induction rate of prothallus and sporophyte of Cibotium barometz. This method can promote the rapid germination of Cibotium barometz spores and improve the induction rate of prothallus and sporophyte. Background Technology

[0002] *Cibotium barometz* (L.) J.Sm. is a perennial tree-shaped fern belonging to the genus *Cibotium*. It is a Class II protected plant in my country and an important raw material for traditional Chinese medicine (listed as "Gouji" in the Chinese Pharmacopoeia). While *Cibotium barometz* is a traditional medicinal plant with unique ornamental value, the artificially propagated spores cannot meet market demand, leading to severe habitat destruction and endangering wild populations. Furthermore, the low natural reproduction rate of spores has resulted in a sharp decline in wild *Cibotium barometz* numbers. Preliminary research has been conducted on tissue culture seedling technology, but this method requires advanced equipment and skilled personnel, increasing seedling costs. Therefore, developing a highly efficient soil-seeding seedling technology for *Cibotium barometz* is of great significance for its protection and future development and utilization.

[0003] The spore propagation of *Cibotium barometz* requires specific environmental conditions. Soil, temperature, humidity, and light all affect spore reproduction. Unsuitable conditions result in low spore germination rates, low prothallium and sporophyte formation rates, and slow development. Currently, some scholars have conducted research on soil-seeding seedling cultivation techniques for *Cibotium barometz*. For example, Chinese patent application CN119522821A, entitled "A Method for Soil-Seeding Seedlings of *Cibotium barometz*", uses peat moss as a substrate, sowing *Cibotium barometz* spores into the substrate and cultivating them until sporophyte seedlings are obtained. Specifically, after sowing, the substrate needs to be moistened to maintain a moisture content of 85-95%. Nutrient solution is sprayed onto the gametophytes starting 45 days after sowing, once every 5 days for a total of 6 sprays. This method is cumbersome and labor-intensive. Chinese patent application CN116569796A, entitled "A Method for Propagating Spores of *Cibotium barometz*", requires the seedling substrate to be fully decomposed, fermented, and crushed, with a minimum seedling time of 152 days (when seedlings reach a height of 6-10 cm). Chinese patent application CN117694188A, entitled "A Method for Propagating Spore Seedlings of *Cibotium barometz*", involves sterilizing the seedling substrate, adding an inducer to the suspension, and spraying the suspension onto the substrate for cultivation. However, this method does not mention the sterilization method or whether the substrate moisture content needs to be maintained. These methods involve numerous procedures, and some lack complete data on the impact of external conditions on spore reproduction. Summary of the Invention

[0004] This invention aims to provide a method for improving the induction rate of prothallus and sporophyte of Cibotium barometz. This method can promote the rapid germination of Cibotium barometz spores and improve the induction rate of prothallus and sporophyte. It has the advantages of simple operation, time and labor saving, and high induction rate.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for improving the induction rate of prothallus and sporophyte of Cibotium barometz includes the following steps:

[0007] S1. The substrate is sterilized with a bactericide, including thiophanate-methyl and tebuconazole. After sterilization, the moisture content of the substrate is reduced to 40-50%, and the sterilized substrate is obtained.

[0008] S2. Bring the bottom of the substrate after sterilization in step S1 into contact with the hormone aqueous solution to obtain a substrate that has absorbed the hormone aqueous solution.

[0009] S3. Spread the golden bream spores on the surface of the substrate containing the hormone solution in step S2 and culture them.

[0010] Treatments 19 to 27 in the screening experiments of this invention involved filling containers with the substrate and spreading it out. A mixture of thiophanate-methyl, azoxystrobin, and tebuconazole was then used to thoroughly soak the substrate before sowing and cultivation. The results showed that these nine treatments not only had high contamination rates, but also prolonged the induction time of uncontaminated prothalliums to 40–63 days. As mold multiplied and infected the prothalliums, they became extremely susceptible to death.

[0011] This invention makes creative adjustments to this, employing the following three key technical points interacting with each other: (1) The seedling substrate is fully soaked in a fungicide to ensure that the seedling process is not contaminated (the fungicide thiophanate-methyl and azoxystrobin•tebuconazole can synergistically kill bacteria, ensuring that the substrate will not grow mold); (2) The moisture content of the fungicide substrate is reduced to 40-50%, at which point the substrate is relatively loose, making the substrate flat and allowing the spores to receive sufficient light (the control group that is only fungicided but not air-dried will have a high contamination rate, prolonged prothallium induction time, and prothallium is easily infected by mold); (3) The bottom of the substrate continuously absorbs hormone aqueous solution, providing nutrients and water for rapid spore germination and prothallium induction. The above three key technical points interact with each other to improve the prothallium and sporophyte induction rate.

[0012] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0013] In step S1, the bactericide is a mixture of 800-1000 times dilution of thiophanate-methyl with an effective ingredient content of 60-80% and 1500-2000 times dilution of tebuconazole with an effective ingredient content of 20-40%. 3-5 parts by volume of the bactericide are mixed with 1 part by volume of the matrix.

[0014] The active ingredient in thiophanate-methyl is thiophanate (chemical name: 1,2-bis(3-methoxycarbonyl-2-thiourea)benzene). The active ingredients in pyraclostrobin and tebuconazole are pyraclostrobin and tebuconazole.

[0015] In one preferred embodiment, in step S2, the aqueous hormone solution comprises 6-benzylaminopurine and indoleacetic acid.

[0016] Preferably, each 1000 ml of the hormone aqueous solution contains 0.08–0.10 mg of 6-benzylaminopurine and 0.10–0.15 mg of indoleacetic acid.

[0017] In one preferred embodiment, in step S2, the substrate sterilized in step S1 is placed in a seedling container, the bottom of which has a slit, the length of which is 2 / 3 to 3 / 4 of the length of the seedling container.

[0018] In one preferred embodiment, in step S2, the seedling container is a square box with a height of 4.0 to 5.0 cm, the substrate height is 1 / 4 to 1 / 3 of the box height, and the substrate surface is flat.

[0019] In one preferred embodiment, in step S2, the bottom of the seedling container is immersed in a hormone solution, the depth of which is 2.0-3.0 cm, and the immersion time is 10-12 hours.

[0020] In one preferred embodiment, in step S3, after the golden bream spores are sown on the surface of the substrate containing the hormone aqueous solution in step S2, the lid is placed on and sealed, and the substrate is cultured for 35-40 days.

[0021] Preferably, a plastic wrap is used to seal the lid. The water solution soaked in the bottom of the plastic container and the sealed plastic wrap provide nutrients and moisture for rapid spore germination and protothallus induction.

[0022] In one preferred embodiment, in step S3, after culturing for 35-40 days, the seedling container is left unsealed without opening the lid. The container is then soaked in sterile water at a depth of 2.0-3.0 cm for 20-24 hours. After soaking, the seedling container is sealed.

[0023] After a certain period of cultivation, the sporophyte is induced by soaking the bottom of the container in sterile water and sealing it with plastic wrap.

[0024] In one preferred embodiment, in step S3, the culture room temperature is 25–30°C, the light intensity is 2500–4500 lux, and the light duration is 12–14 h / d. Temperatures that are too high or too low are detrimental to spore germination and subsequent growth and development. A light intensity of 3500–4500 lux results in a shorter induction time for prothallium and sporophyte formation.

[0025] Preferably, in step S3, the light intensity is 3500–4500 lux.

[0026] In one preferred embodiment, the seeding rate in step S3 is (0.03~0.04) g / m³. 2 .

[0027] In step S1, after sterilization, the product is naturally air-dried until the moisture content is 40-50%.

[0028] In step S1, the substrate includes peat moss and sphagnum moss. Preferably, the substrate includes 1 to 4 parts by volume of peat moss and 1 to 2 parts by volume of sphagnum moss.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention identifies a low-cost yet highly effective sterilization method. By improving sowing conditions and methods, spores are sown onto a smooth substrate surface. Moisture is replenished promptly from the bottom of the substrate. This combination of key factors ultimately improves the induction rates of prothalliums and sporophytes. Using this method to cultivate *Cibotium barometz* seedlings, prothalliums were induced in an average of 22 days, and sporophytes in 66 days. This method can be widely applied to *Cibotium barometz* sowing and seedling cultivation, saving seedling time and labor costs.

[0031] 1. This invention uses a suitable mixed bactericide to thoroughly kill harmful bacteria in the substrate by soaking it for a long time, and no contaminants are generated during the cultivation process. Compared with high temperature and high pressure sterilization, this invention reduces production costs, improves market competitiveness, and is suitable for factory production.

[0032] 2. After sterilization, the substrate of this invention is naturally air-dried to a moisture content of 40-50%. At this point, the substrate is loose and its surface is flat when sprinkled in a container. By soaking the bottom in an aqueous solution containing hormones, the substrate absorbs sufficient water, and the spores are distributed on the flat substrate surface. This ensures that the rhizoids of the prothallus can fully absorb nutrients and water, and also receive maximum light, which is beneficial for spore germination and prothallus induction.

[0033] 3. After 35-40 days of cultivation, a large number of prothalliums are formed. At this time, water is replenished by soaking in sterile water. In addition, the container is sealed, which provides conditions for the development of sperm and eggs on the prothalliums and fertilization in the later stage, thereby improving the sporophyte induction rate.

[0034] Using this method, prothallus (visible to the naked eye) is induced after an average of 22 days of culture, and microsporophyte is induced after 66 days. Both are bright green in color, and the seedlings emerge uniformly. The culture cycle is significantly shortened. This invention can be applied on a large scale to the sowing and seedling cultivation of Golden Retriever. Attached Figure Description

[0035] Figure 1 The protophylls (40d) induced by treatment 6 in this embodiment of the invention.

[0036] Figure 2 The sporophyte (90d) induced by treatment 6 in this embodiment of the invention. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0038] The embodiments described herein are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0039] Example 1:

[0040] This invention uses a mixture of: ① a substrate consisting of 2 parts by volume of Northeast peat soil (purchased from Lvwo Nursery in Hengdaohe Town, with moderate decomposition, loose and breathable, pH=5.5-6.0; organic matter content >60%, total humic acid content >35%, free humic acid >30%, total nitrogen content >1.5%, total phosphorus content >0.1%; cellulose content accounting for more than 50% of organic matter) and 1 part by volume of imported peat soil (purchased from JIFFY in the Netherlands, particle size ≤20mm); ② a square transparent plastic box, 5.0cm high, with a slit cut at the bottom, the length of which is 3 / 4 of the box length; ③ an aqueous solution containing 0.10mg of 6-benzylaminopurine and 0.15mg of indoleacetic acid per 1000ml, with the remainder being water; ④ spores are evenly sown on the substrate surface (0.04g / m²). 2 Cover the plastic box with the lid, seal the plastic box with plastic wrap, and incubate under specific temperature and light conditions. ⑤ After 40 days of incubation, remove the plastic wrap, without opening the plastic box lid, immerse the box in sterile water, seal the container with new plastic wrap, and incubate under specific temperature and light conditions.

[0041] Different sterilization methods are used for treatment:

[0042] (A) Soak the substrate in ① above in advance with a fungicide mixture of "thiophanate-methyl + azoxystrobin·tebuconazole". The fungicide is a mixture of 800 times dilution of thiophanate-methyl (70% active ingredient by mass) and 1500 times dilution of azoxystrobin·tebuconazole (30% active ingredient by mass). Mix 5 parts by volume of the fungicide mixture with 1 part by volume of the substrate. Allow it to air dry naturally until the moisture content is 50%. Spread the substrate in the container in ② above, ensuring the surface is flat. Before sowing, soak the container in the aqueous solution in ③ above for 10 hours. Sow according to the spore quantity and method in ④ above to induce prothallium. Induce sporophytes according to the method in ⑤ above.

[0043] (B) Using 1.1 kg / cm 2 The above-mentioned seedling substrate was sterilized at 121℃ under high pressure for 25 minutes, and then the substrate was placed into the container described in step ②. Before sowing, the container was soaked in the aqueous solution described in step ③ for 10 hours. Sowing was carried out according to the spore quantity and method described in step ④ to induce prothallus. Sporophyte induction was carried out according to the method described in step ⑤.

[0044] (C) Place the above-mentioned substrate ① into a container and spread it evenly. Thoroughly drench the substrate with a mixture of "thiophanate-methyl + azoxystrobin·tebuconazole". The fungicide is a mixture of 800 times dilution of thiophanate-methyl (70% active ingredient by mass) and 1500 times dilution of azoxystrobin·tebuconazole (30% active ingredient by mass). Mix 5 parts by volume of the fungicide mixture with 1 part by volume of the substrate. Sow according to the spore quantity and method described in ④ above to induce prothallus. Induce sporophytes according to the method described in ⑤ above.

[0045] Test temperatures: (α) 20~24℃; (β) 25~30℃; (γ) 31~35℃.

[0046] Experimental illumination: (I) 0; (II) 2500~3500 lux; (III) 3500~4500 lux, illumination duration 12h / d.

[0047] A randomized block design was used, with each treatment replicated three times, and 10 seedling boxes sown per replicate. Prothallus germination time: visible green dots. Sporophyte germination time: visible sporophytes. Contamination rate: calculated based on visible mold coverage. Prothallus induction rate: calculated at 80 days after sowing, based on prothallus coverage in the seedling container, regardless of prothallus maturity; Sporophyte induction rate: calculated at 120 days after sowing, based on sporophyte coverage in the seedling container, regardless of sporophyte size.

[0048]

[0049]

[0050] As shown in Table 1-2, the thoroughness of substrate sterilization and moisture content are crucial during the sowing and seedling raising process of Golden Retrievers.

[0051] Treatments 1-9 involved pre-soaking the substrate in a mixture of thiophanate-methyl, azoxystrobin, and tebuconazole, followed by natural air drying to a moisture content of 40-50%. Before sowing, the substrate was soaked in the solution at the bottom of the container. After sowing, the container was sealed with plastic wrap, and later, the plastic wrap was opened and the substrate was soaked in sterile water. No mold grew in any of these treatments. Treatments 10-18 showed mold growth in the substrate starting on the second day after sowing. Within five days, the mold covered the entire container, ultimately preventing spore germination. Treatments 19-27 showed a small amount of mold growth in the substrate 20 days after sowing, with a contamination rate as high as 70.0-86.7% after 100 days. As the mold increased, the induced prothallus and sporophyte were also largely infected and died.

[0052] A smooth seedling substrate is also beneficial for spore germination and growth. Because the substrates for treatments 1-9 were naturally air-dried, they were loose in the containers and did not lose their smoothness after soaking in water. This allowed spores to obtain sufficient nutrients, water, and light, resulting in rapid spore germination. The prothallus induction time was 22-53 days, with a maximum induction rate of 93.2%; the sporophyte induction time was 66-93 days, with a maximum induction rate of 89.9%. Treatments 19-27 involved filling containers with the substrate and leveling it, then drenching the substrate with a mixture of thiophanate-methyl, azoxystrobin, and tebuconazole before sowing and cultivation. The result was that these nine treatments not only had a high contamination rate, but the induction time for uncontaminated prothallus was prolonged to 40-63 days. With the proliferation and infection of mold, the prothallus were highly susceptible to death.

[0053] Light and temperature are also crucial for spore germination and development. Results from treatments 1, 4, 7, 19, 22, and 25 showed that *Cibotium barometz* spores would not germinate without light. Although the induction rates were similar for treatments 5 (2500–3500 lux) and 6 (3500–4500 lux), the induction times for prothalliums and sporophytes in treatment 5 were on average 10 and 14 days later than in treatment 6, respectively. Temperatures that were too high or too low were detrimental to spore germination and later growth. Treatments 3, 6, and 9 were at temperatures of 20–24℃, 25–30℃, and 31–35℃, respectively, with prothallium germination times of 30 days, 22 days, and 53 days, and induction rates of 88.7%, 93.2%, and 45.0%, respectively. The sporophyte germination times were 75 days, 66 days, and 93 days, with induction rates of 83.8%, 89.9%, and 43.2%, respectively. Therefore, the optimal conditions for culturing *Cyprinus pubescens* are 3500–4500 lux of light and 25–30℃, under which the induction rates of prothallium and sporophyte are high.

[0054] Soil-seeding of *Cephalotaxus fortunei* is an open-type seedling cultivation method. The seedling environment has high humidity (generally above 95%), and the substrate is highly susceptible to mold infection under high temperature and humidity conditions; even a small amount of mold can multiply rapidly. Therefore, the thoroughness of substrate sterilization, spore exposure to light, and sufficient moisture content during cultivation are all crucial for spore germination, prothallus and sporophyte induction. Using the fungicide and treatment method of this invention, no mold was generated during the seedling cultivation process. Simultaneously, the substrate surface treated by this invention is smooth, ensuring sufficient light, nutrients, and moisture for spore germination and growth. After a large number of prothallus are formed, the water required for growth and development is replenished by immersing sterile water at the bottom of the container. With suitable temperature and light, the prothallus induction rate for treatments 5 and 6 is ≥91.5%, and the sporophyte induction rate is ≥85.2%.

[0055] Comparative Example 1

[0056] The difference between this comparative example and treatment 6 in Example 1 is that it does not include tebuconazole and thiophanate-methyl, but instead uses thiophanate-methyl to soak the above-mentioned substrate in advance, air-dry it naturally to a moisture content of 40-50%, and then sow it according to the container and method described in this invention, and cultivate it under the conditions of temperature 25-30°C and light intensity 3500-4500 lux.

[0057] As the spore culture time increased, the amount of mold gradually increased. The average contamination rate was 20.3% after 120 days of sowing. Prothallium was induced after 25 days, with an induction rate of 83.3%; sporophyte induction time was 70 days, with an induction rate of 75.5%. The experimental results show that the contamination rate of this comparative example is higher than that of the treatment with the mixture of "thiophanate-methyl + azoxystrobin·tebuconazole", and the induction rates of both prothallium and sporophyte are lower than those of treatment 6 in Example 1.

[0058] Comparative Example 2

[0059] The difference between this comparative example and treatment 6 in Example 1 is that it does not include thiophanate-methyl, but instead uses tebuconazole and azoxystrobin to soak the above-mentioned substrate in advance, and then air-dry it naturally until the moisture content is 40-50%. It is then sown according to the container and method described in this invention and cultured under the conditions of temperature 25-30°C and light intensity 3500-4500 lux.

[0060] As spore germination time increased, mold gradually increased. The average contamination rate was 16.5% after 120 days of sowing. Prothallium was induced at 25 days, with an induction rate of 75.7%; sporophyte induction time was 73 days, with an induction rate of 68.9%. The experimental results show that the contamination rate of this comparative example is higher than that of soaking in the mixture of "thiophanate-methyl + azoxystrobin·tebuconazole", and the induction rates of both prothallium and sporophyte are lower than those of treatment 6 in Example 1.

[0061] Comparative Example 3

[0062] The difference between this comparative example and treatment 6 in Example 1 is that the substrate was soaked in a mixture of "thiophanate-methyl + tebuconazole" and placed in a container before it was naturally air-dried (the substrate was squeezed tightly and there was water between the fingers). It was not soaked in the above-mentioned aqueous solution (each 1000 ml of the hormone aqueous solution contains 0.08-0.10 mg of 6-benzylaminopurine and 0.10-0.15 mg of indoleacetic acid). After direct sowing, the box was sealed with plastic wrap and cultured under the conditions of 25-30°C and 3500-4500 lux of light.

[0063] No mold developed in the golden retriever cultured under these conditions. The prothallus induction time was 28 days, with an induction rate of 70.2%; the sporophyte induction time was 70 days, with an induction rate of 65.2%. Compared with treatment 6 in Example 1, the induction time for prothallus and sporophyte in this comparative example was basically the same, but the induction rate was lower.

[0064] Comparative Example 4

[0065] The difference between this comparative example and treatment 6 in Example 1 is that the substrate was not soaked in the above-mentioned aqueous solution (each 1000 ml of the hormone aqueous solution contains 0.08-0.10 mg of 6-benzylaminopurine and 0.10-0.15 mg of indoleacetic acid) before sowing. Instead, the spores were directly sown on the substrate with a moisture content of 40-50%. After sowing, the box was sealed with plastic wrap and cultured under the conditions of 25-30°C and 3500-4500 lux of light.

[0066] After 40 days of sowing, no prothalliums were induced. The plastic wrap was removed, but the lid was not opened. The container was then immersed in sterile water, and after soaking, the plastic container was sealed with plastic wrap and cultured for another 60 days. Prothalliums were induced after 60 days, and the prothallium induction rate was 71.4% after 100 days. Both the induction time and the induction rate were lower than those of treatment 6 in Example 1.

[0067] Comparative Example 5

[0068] The difference between this comparative example and treatment 6 in Example 1 is that the above aqueous solution does not contain 0.08-0.10 mg of 6-benzylaminopurine or 0.10-0.15 mg of indoleacetic acid (i.e., it is pure water without any added hormones). Other treatments are the same, and the samples are cultured at a temperature of 25-30°C and a light intensity of 3500-4500 lux.

[0069] The induction time for prothallium and sporophyte was basically the same as that of treatment 6 in Example 1, but the induction rates at 80 days after sowing were 88.8% and 73.2%, respectively, which were lower than those of treatment 6.

Claims

1. A method for improving the induction rate of prothallus and sporophyte in *Cibotium barometz*, comprising the following steps: S1. Sterilize the substrate with a bactericide, the bactericide including thiophanate-methyl and tebuconazole. After sterilization, reduce the moisture content of the substrate to 40-50%. Spread the substrate in a container, ensuring the surface is flat, to obtain the sterilized substrate. S2. The bottom of the substrate sterilized in step S1 is brought into contact with the hormone aqueous solution for 10-12 hours to obtain a substrate that has absorbed the hormone aqueous solution. In step S2, the hormone aqueous solution comprises 6-benzylaminopurine and indoleacetic acid; S3. Spread the golden dog spores on the substrate surface containing the hormone solution in step S2 and culture them. In step S3, after the golden dog spores are sown on the surface of the substrate containing the hormone solution in step S2, the lid is covered and sealed, and cultured for 35-40 days. In step S3, after culturing for 35-40 days, keep the seedling container unsealed and do not open the lid. Soak the container in sterile water to a depth of 2.0-3.0 cm for 20-24 hours. After soaking, seal the seedling container.

2. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 1, characterized in that, In step S1, the bactericide is a mixture of 800-1000 times dilution of thiophanate-methyl with an effective ingredient content of 60-80% and 1500-2000 times dilution of tebuconazole with an effective ingredient content of 20-40%. 3-5 parts by volume of the bactericide are mixed with 1 part by volume of the matrix.

3. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 1, characterized in that, In step S2, the substrate sterilized in step S1 is placed in a seedling container. The bottom of the seedling container has a slit, and the length of the slit is 2 / 3 to 3 / 4 of the length of the seedling container.

4. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 3, characterized in that, In step S2, the seedling container is a square box with a height of 4.0 to 5.0 cm, the substrate height is 1 / 4 to 1 / 3 of the box height, and the substrate surface is flat.

5. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 3, characterized in that, In step S2, the bottom of the seedling container is immersed in a hormone solution, with the hormone solution reaching a depth of 2.0 to 3.0 cm.

6. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to any one of claims 1-5, characterized in that, In step S3, the culture room temperature is 25-30℃, the light intensity is 2500-4500 lux, and the light duration is 12-14 h / d.

7. The method for improving the induction rate of prothallus and sporophyte of *Cibotium barometz* according to any one of claims 1-5, characterized in that, The seeding rate in step S3 is (0.03~0.04) g / m². 2 .

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